Field of the Invention (Technical Field)
The present invention relates to the use of the sucrose phosphate synthase (SPS) gene of maize, and closely related regulatory genes, for altering the biosynthesis and accumulation of sucrose in alfalfa and other legumes. The present invention further relates to transgenic constructs containing the SPS and allied regulatory genes, for use in the transformation of alfalfa and other legumes, and it also relates to transgenic plants containing such constructs. The present invention also relates to the use of the glutamine synthetase (GS) gene of soybean, and closely related regulatory genes, for altering the assimilation of nitrogen in alfalfa and other legumes. The present invention further relates to transgenic constructs containing the GS1 gene and allied regulatory genes, for use in the transformation of alfalfa and other legumes, and it also relates to transgenic plants containing such constructs.
Description of Related Art
Sucrose phosphate synthase (SPS) is an enzyme used in the synthesis of sucrose in plants. SPS is involved in the synthesis of sucrose by the transfer of glucosyl moiety from UDP-glucose to fructose-6-phosphate, which is dephosphorylated by the action of sucrose-6-phosphate phosphatase (SPP) to yield sucrose. Sucrose is a stable product of photosynthesis that is transported from the photosynthetic tissues via the phloem into heterotrophic tissues which includes the root nodules in leguminous plants. The root nodule is an organ formed as a result of a symbiotic association of legume plants with a soil bacteria, Rhizobium. The root nodule is the site where the symbiont can convert free nitrogen into NH3, which is then assimilated by host encoded enzymes. The symbiont is contained in a membrane bound vesicle called the symbiosome and this membrane is called the peribacteroid membrane which contains many host encoded proteins. The enzyme nitrogenase in the bacteria (symbiont), catalyzes ATP dependent reduction of N2 to NH3 in infected cells of the root nodules. Any ammonium produced is exported out through the peribacteroid membrane into the cytosol, where it is assimilated via host encoded enzymes glutamine synthetase (GS) and glutamate synthase to produce glutamine and glutamate.
The nodules are primarily dependent on the import and metabolism of sucrose to fuel the N2 fixation process. Sucrose is metabolized initially by sucrose synthase (SuSy) (and to a lesser extent vacuolar invertase) and thereafter, via glycolysis to phosphoenolpyruvate (PEP). The products of sucrose metabolism have two major functions in the nodules. The first is to provide a substrate that can cross the peribacteroid membrane and be oxidized to provide the ATP and reducing power for the fixation of N2. The second is its involvement in the assimilation of ammonia which is produced by the bacteria, the synthesis of nitrogen products and their export from the nodule via the xylem. The high carbon cost for the alfalfa/Rhizobium interaction renders the nodule a strong sink for sucrose. Furthermore, sucrose is stored in high concentrations in nodules during the photoperiod and utilized in the dark period (at night).
Sucrose plays a role in the nodules. SPS is encoded by a small multigene family. The family members, besides showing differences in tissue-specific expression, are also subject to differential regulation at the posttranslational level via phosphorylation. In alfalfa, SPSB gene shows leaf-specific expression and SPSA gene, though nodule-enhanced, exhibits constitutive expression. Embodiments of the present invention comprise genetically engineered alfalfa that expresses a maize SPSB gene driven by the CaMV35S promoter. These transformants show increased nodulation and N2-fixation and overall increased N content at the whole plant level compared to control alfalfa plants. Moreover, the transformants show late flowering, shorter internodes, and intense green coloration mimicking the phenotypes seen in super/hypernodulating legumes.
Glutamine synthetase (GS) plays a central role in nitrogen metabolism in all plants. GS catalyzes the ATP dependent condensation of ammonia with glutamate, to yield glutamine. Plant GS is an octamer and occurs as a number of isoenzyme forms and these GS isoforms are located either in the cytosol (GS1) or chloroplast/plastid (GS2). GS1 represents a key component of nitrogen use efficiency and yield. Increase in GS1 activity is accompanied by a substantial improvement in plant performance.
Regulation of expression of a soybean (Glycine max) GS1 gene (Gmglnβ1) driven by a constitutive promoter (CaMV 35S) in transgenic plants typically occurs at a post-transcriptional level. The post-transcriptional regulatory step of the Gmglnβ1 and also the GS1 genes from alfalfa, is at the level of transcript turnover, mediated by their 3′UTR. Gmglnβ1 was investigated to determine if it is also subject to other modes of regulation by testing the role of its 5′UTR in the regulation of gene expression.
The synthesis of glutamine is a first step for the synthesis of all other essential nitrogenous compounds contained in the cells. GS also plays a crucial role in removing ammonia which is toxic to the cells, but at the same time GS levels and activity have to be fine-tuned to maintain a balance between the rates of amino acid biosynthesis and the cellular C skeletons and ATP levels, since they can be depleted by GS activity. GS in bacteria is highly regulated in vivo by transcriptional and post-translational mechanisms including adenylylation and metabolic feedback inhibition. The glutamine/α-ketoglutarate ratio and the adenylate energy charge are critical in the control of GS activity in bacteria. The glutamine/α-ketoglutarate ratio is also important for the control of nitrogen assimilation in plants. GS in plants, as in bacteria, is also regulated at multiple levels.
Analysis of the translation process of eukaryotic mRNAs has shown that the 5′UTR plays an important role in translation initiation by its secondary structure; the context of AUG codon; and the existence of AUG or upstream open-reading frames. The 5′UTR is also the target for the binding of microRNAs that cause translational repression or enhance translation. There are also some reports of the 5′UTR having a role in mRNA stabilization, though this is usually an attribute of sequences in the 3′UTR.
To demonstrate the role of the 5′UTR of the Gmglnβ1 gene in the regulation of its expression, a series of gene constructs were made with the Gmglnβ1 driven by the CaMV 35S promoter, with and without the 5′ and 3′ UTRs and tested for their expression by agroinfiltration in tobacco (Nicotiana tabacum cv Xanthi) leaves, at both the transcript and protein levels. Transient expression through agroinfiltration is a relatively easy procedure known to be effective in analyzing expression of transgenes. Results showed that whereas the 3′UTR of the GS1 gene is involved in the control of the mRNA accumulation or stability, the Gmglnβ1 5′UTR enhances the translation of both the GS1 gene and a β-glucuronidase (uidA) reporter gene in plants.
For a complete characterization of the 5′UTR of the Gmglnβ1 gene and its role as a translation enhancer, the question of whether the 5′UTR of Gmglnβ1 would have the necessary information for allowing initiation of translation in a bacterial cell was also addressed. In the traditional view of translation initiation, there are major differences between eukaryotes and prokaryotes in the way that ribosomes are recruited to the mRNA and this is mediated by sequences in the 5′ non-coding region. However, there are reports that eukaryotic ribosomes can recognize prokaryotic signals and initiate synthesis at internal sites of polycistronic mRNAs. Similarly, the Escherichia coli ribosomes have been shown to recognize eukaryotic viral initiation signals and translate eukaryotic viral mRNAs suggesting that translation initiation signals in prokaryotes and eukaryotes are similar. To corroborate the universality of the translation initiation mechanism between prokaryotes and eukaryotes, the Gmglnβ1 gene was introduced with its 5′UTR in E. coli and showed accumulation of the corresponding protein in the bacterial cells, thus supporting the postulate that the mechanisms of translation initiation for this gene are conserved between plants and bacteria and may further support the notion that GS genes may have originated from a gene duplication event that preceded the divergence of prokaryotes and eukaryotes.